Astrocytes are a major type of glia that play critical roles in the development and function of the nervous system. Malfunction of astrocytes are involved in neurological disorders including glioma, autism, amyotrophic lateral sclerosis, traumatic brain injury and stroke. How astrocyte proliferation and differentiation are regulated remains poorly understood. Increased astrocyte proliferation in humans contributes to the expansion in brain size in human evolution, and is potentially important for human intelligence. Unchecked proliferation of astrocytes, however, can lead to glioma. The mechanistic differences in the regulation of astrocyte proliferation and differentiation in humans and mice are unknown. Transcription factors specifically expressed by a cell type are key regulators of cell differentiation. Although transcriptional regulations of astrocytes have been studied in the spinal cord and the retina, the transcription factor(s) that regulate astrocyte proliferation and differentiation in the brain remains elusive. In my preliminary studies, I used innovative methods to purify all of the major cell types from mouse brains and obtained sensitive and accurate transcriptome datasets of each of the cell types by RNA-sequencing. I identified three astrocyte-specific transcription factors with this unbiased approach. Mice deficient for one of these factors have substantially reduced expression of astrocyte genes. In addition, I developed the first method to acutely purify astrocytes and their progenitors from human brains and I optimized a culturing condition that prevents these astrocytes from becoming reactive, which is a major limitation of existing methods. Building on these results, I propose to test the hypothesis that th three astrocyte-specific transcription factors are necessary and sufficient for astrocytes differentiation and that the differential regulation of these factors underlies the increase of astrocytes in human brains compared with mouse brains. In the K99 phase, I will test the necessity and sufficiency of these transcription factors in astrocyte proliferation and differentiation using existing knockout mouse lines, and a combination of in vitro and in vivo molecular manipulation techniques including viral infection and in utero electroporation. I will acquire expertise in molecular manipulations from the mentoring labs. I will also examine the regulatory interactions between these three transcription factors and determine whether a transcriptional cascade formed by the three factors sequentially regulate astrocyte specification, proliferation, and maturation. Finally, as an independent investigator, I will utilize K99 phase training in molecular manipulations and examine the role of the three transcription factors in human astrocyte development with the new purification and culturing method I developed. I will also investigate the mechanisms underlying the increase of astrocytes in humans. The proposed research is expected to close a major knowledge gap in brain development, as astrocytes are the last major cell type of the brain without knowledge of the transcriptional regulation of their differentiation. Moreover, knowledge obtained from this project has the potential to advance the treatment of glioma.